The present invention relates generally to the inhibition of blood clotting proteins and more particularly to specific inhibitors of the blood clotting enzyme factor Xa.
The ability to form blood clots is vital to survival. In certain disease states, however, the formation of blood clots within the circulatory system is itself a source of morbidity. Thus, it sometimes can be desirable to prevent blood clot formation. However, it is not desirable to completely inhibit the clotting system because life threatening hemorrhage would ensue.
In order to reduce the intravascular formation of blood clots, those skilled in the art have endeavored to develop an effective inhibitor of prothrombinase or of factor Xa, which is incorporated into the prothrombinase complex where it activates thrombin during clot formation.
Appropriate concentrations of a factor Xa inhibitor would increase the level of prothrombinase forming agents required to initiate clotting but would not unduly prolong the clotting process once a threshold concentration of thrombin had been obtained. However, despite the long standing recognition of the desirability of such an inhibitor, there is at present no effective, specific factor Xa inhibitor in clinical use.
In many clinical applications there is a great need for anti-coagulant treatment. The currently available drugs are not satisfactory in many specific clinical applications. For example, nearly 50% of patients who undergo a total hip replacement develop deep vein thrombosis (DVT). The currently approved therapies include fixed dose low molecular weight heparin (LMWH) and variable dose heparin. Even with these drug regimes, 10% to 20% of patients develop DVT and 5% to 10% develop bleeding complications.
Another clinical situation for which better anti-coagulants are needed concerns subjects undergoing transluminal coronary angioplasty and at risk for myocardial infarction or suffering from crescendo angina. The present, conventionally accepted therapy, which consists of administering heparin and aspirin, is associated with a 6% to 8% abrupt vessel closure rate within 24 hours of the procedure. The rate of bleeding complications requiring transfusion therapy due to the use of heparin also is approximately 7%. Moreover, even though delayed closures are significant, administration of heparin after the termination of the procedures is of little value and can be detrimental.
The most widely used blood-clotting inhibitors are heparin and the related sulfated polysaccharides, LMWH and heparin sulfate. These molecules exert their anti-clotting effects by promoting the binding of a natural regulator of the clotting process, anti-thrombin III, to thrombin and to factor Xa. The inhibitory activity of heparin primarily is directed toward thrombin, which is inactivated approximately 100 times faster than factor Xa. Although relative to heparin, heparin sulfate and LMWH are somewhat more potent inhibitors of Xa than of thrombin, the differences in vitro are modest (3-30 fold) and effects in vivo can be inconsequential. Hirudin and hirulog are two additional thrombin-specific anticoagulants presently in clinical trials. However, these anticoagulants, which inhibit thrombin, also are associated with bleeding complications.
Preclinical studies in baboons and dogs have shown that specific inhibitors of factor Xa prevent clot formation without producing the bleeding side effects observed with direct thrombin inhibitors. Such factor Xa inhibitors include, for example, 2,7-bis-(4-amidino benzylidene)-cycloheptanone and Nxcex1-tosylglycyl-3-amidinophenylalanine methyl ester (xe2x80x9cTENSTOPxe2x80x9d), which have effective inhibitory concentrations (Ki""s) of about 20 nM and 800 nM, respectively. (+)-(2S)-2-(4({(3S)-1-acetimidoyl-3-pyrrolidinyl}oxy)phenyl)-3-(7-amidino-2-naphthyl)propanoic acid also is representative of a class of factor Xa inhibitors (Katakura et al., Biochem. Biophys. Res. Comm. 197:965-972 (1993)). Thus far, however, these compounds have not been developed clinically.
Specific protein inhibitors of factor Xa also have been identified and include, for example, antistasin (xe2x80x9cATSxe2x80x9d) and tick anticoagulant peptide (xe2x80x9cTAPxe2x80x9d). ATS, which isolated from the leech, Haementerin officinalis, contains 119 amino acids and has a Ki for factor Xa of 0.05 nM. TAP, which is isolated from the tick, Ornithodoros moubata, contains 60 amino acids and has a Ki for factor Xa of about 0.5 nM.
The effectiveness of recombinantly-produced ATS and TAP have been investigated in a number of animal model systems. Both inhibitors decrease bleeding time compared to other anticoagulants and prevent clotting in a thromboplastin-induced, ligated jugular vein model of deep vein thrombosis. The results achieved in this model correlate with results obtained using the current drug of choice, heparin.
Subcutaneous ATS also was found to be an effective treatment in a thromboplastin-induced model of disseminated intravascular coagulation (DIC). TAP effectively prevents xe2x80x9chigh-shearxe2x80x9d arterial thrombosis and xe2x80x9creduced flowxe2x80x9d caused by the surgical placement of a polyester (xe2x80x9cDACRONxe2x80x9d) graft at levels that produced a clinically acceptable prolongation of the activated partial thromboplastin time (aPTT), i.e., less than about two fold prolongation. By comparison, standard heparin, even at doses causing a five fold increase in the aPTT, did not prevent thrombosis and reduced flow within the graft. The aPTT is a clinical assay of coagulation which is particularly sensitive to thrombin inhibitors.
ATS and TAP have not been developed clinically. One major disadvantage of these two inhibitors is that administration of the required repeated doses causes the generation of neutralizing antibodies, thus limiting their potential clinical use. Moreover, the sizes of TAP and ATS render oral administration impossible, further restricting the number of patients able to benefit from these agents.
A specific inhibitor of factor Xa would have substantial practical value in the practice of medicine. In particular, a factor Xa inhibitor would be effective under circumstances where the present drugs of choice, heparin and related sulfated polysaccharides, are ineffective or only marginally effective. Thus, there exists a need for a low molecular weight, factor Xa-specific blood clotting inhibitor that is effective, but does not cause unwanted side effects. The present invention satisfies this need and provides related advantages as well.
The present invention provides compounds that specifically inhibit factor Xa activity. A compound of the invention has the structure X1-Y-I-R-X2, wherein X1 is a hydrogen (H), acyl, alkyl or arylalkyl group, or one or more amino acids, and X2 is a modified C-terminal group, one or more carboxy-protecting groups (see below), one or more amino acids, or other substituents, and Y, I and R refer to the amino acids tyrosine, isoleucine and arginine, respectively, and to peptidomimetic or organic structures that have the same functional activities as tyrosine, isoleucine and arginine, respectively. In addition, a compound of the invention has the structure, A1xe2x80x94A2xe2x80x94(A3)mxe2x80x94B, as defined herein.
A compound of the invention can be linear or cyclic, between about 2 and 43 residues in length and modified at the N-terminus or C-terminus or both. Such compounds exhibit a specific inhibition of factor Xa activity with a Kixe2x89xa6100 xcexcM, preferably a Kixe2x89xa62 nM, and do not substantially inhibit the activity of other proteases involved in the coagulation cascade. Specific examples of such compounds include Ac-Tyr-Ile-Arg-Leu-Ala-NH2(SEQ ID NO: 1); Ac-Tyr-Ile-Arg-Leu-Pro-NH2(SEQ ID NO: 2); Ac-(iBu)Tyr-Ile-Arg-Leu-Pro-NH2(SEQ ID NO: 3); Ac-Tyr-Ile-Arg-N(CH3)O(CH3); Ac-Tyr-{xcexa8(CH2NH)}-Ile-Arg-Leu-Pro-NH2(SEQ ID NO: 4) (where xe2x80x9cxcexa8xe2x80x9d indicates a pseudo peptide bond, which, for example, can be a reduced bond as indicated by xe2x80x9c(CH2NH)xe2x80x9d; pseudo peptide bonds are indicated by xe2x80x9cxcexa8xe2x80x9d enclosed in brackets, xe2x80x9c{xcexa8}xe2x80x9d); Ac-Tyr-Ile-Arg-NH-CH2(4-Pyridyl); Ac-Tyr-Ile-{xcexa8(CH2NH)}-Arg-Leu-Pro-NH2(SEQ ID NO: 5); Ac-Tyr-Chg-Arg(NO2)-{xcexa8(CH2NH)}-Leu-NH2; Ac-Tyr-Ile-Arg-{xcexa8(COCH2)}-Gly-Pro-NH2(SEQ ID NO: 6); Ac-Tyr-Ile-Dab(Nxcex3xe2x80x94C3H7N)-Leu-Ala-NH2(SEQ ID NO: 7); Ac-Tyr-Ile-PalMe(3)-NH2; Tyr-Ile-Arg-NH2; (D)-Tyr-Ile-Arg-Leu-Pro-NH2; Ac-(Bzl)Gly-(Chx)Gly-(3-guanidopropyl)Gly-NH2; Cyclo(Gly-Tyr-Ile-Arg-Gly) (SEQ ID NO: 8); Tfa-(iBu)Tyr-Chg-Arg-Leu-Pro-NH2; Ac-pAph-Chg-Arg-Leu-Pro-NH2; Ac-Nal(2)-Chg-Arg-Leu-Pro-NH2; Ac-pAph-Chg-PalMe(3)-NH2; and pharmaceutically acceptable salts and C-terminal derivatives such as amides, esters, alcohols and aldehydes thereof (see, also, Table 5). Methods of specifically inhibiting the activity of factor Xa and of inhibiting blood-clotting in an individual also are provided. Methods of detecting factor Xa levels or activity are provided as well.